Apolipoprotein E (ApoE) is a protein found in the bloodstream in association with a variety of cholesterol and lipid-containing particles. The role of ApoE in metabolism has been reviewed by R. W. Mahley Science, 240: 622-630 (1988), and the complete amino acid sequence of human ApoE has been determined by Rall et al., J. Biol. Chem. 257(8): 4171-4178 (1982).
Through its ability to mediate lipoprotein binding and uptake by lipoprotein receptors, i.e. the LDL receptor and the chylomicron receptor, plasmatic ApoE (p-ApoE) has important functions in the regulation of plasma lipoprotein metabolism and in the maintenance of cholesterol homeostasis. Extensive epidemiological studies have strongly indicated a correlation of high blood-cholesterol levels to heart attacks and strokes, due to the formation of atherosclerotic plaques, the production of which is influenced by both genetic and environmental factors (M. S. Brown and J. L. Goldstein, Science 232: 34 (1986)). Administration of exogenous ApoE may assist in the regulation of serum lipid and cholesterol levels, and in the prevention of atherosclerosis.
In addition, ApoE is produced at a high level and accumulates in the region of injured and regenerating peripheral nerves (M. J. Ignatius et al., Proc. Natl. Acad. Sci. U.S.A. 83: 1125 (1986); G. J. Snipes et al., Proc. Natl. Acad. Sci. U.S.A. 83: 1130; P. A. Dawson et al., J. Biol. Chem. 261: 5681 (1986)). ApoE may be involved in the mobilization and possible reutilization of lipid or the repair, growth and maintenance of myelin and axonal membranes (T. Vogel et al. Proc. Natl. Acad. Sci. U.S.A. 82: 8696 (1985)).
Apolipoproteins including ApoE have also been shown to have immunoregulatory activity (R. W. Mahley et al., J. Lipid Res. 25: 1277 (1984); R. W. Mahley & T. L. Innerarity, Biochem. Biophys. Acta 737, 197 (1983)). ApoE-containing lipoproteins as well as low density lipoproteins have the capacity to inhibit or stimulate antigen-induced and mitogen-induced T lymphocyte activation and proliferation (R. W. Mahley, Science 240: 622-630 (1988)).
However, it is impossible to obtain from human blood sufficient quantities of naturally-occurring ApoE to examine the beneficial therapeutic effects which may be associated with ApoE. Accordingly, there is a need to provide a practical means of producing sufficient quantities of highly purified ApoE to conduct animal and clinical trials and for widespread pharmaceutical use.
The subject invention provides a novel purification method for producing large quantities of highly purified, biologically active, recombinant apolipoprotein E (r-ApoE). This method involves several novel features to solve inter alia problems connected with (1) aggregation and degradation of ApoE, (2) separation of active and inactive forms of ApoE, and (3) removal of endotoxins.
European Patent Publication EP 205,715 assigned to Mitsubishi Chemical Industries Ltd. disclosed the cloning of ApoE in E. coli yeast and CHO cells. However, the disclosure indicated that only minute amounts of ApoE were produced and did not include a purification method.
PCT Publication WO 87 02061 assigned to Biotechnology Research Partners Ltd. disclosed the expression of fusion proteins containing the receptor recognition domain of ApoE for drug delivery. This publication does not disclose a method for production or purification of the full length ApoE analog protein.
PCT Publication WO 87 02059 also assigned to Biotechnology Research Partners Ltd. disclosed the correlation of polymorphisms in apolipoproteins (including ApoE) with atherosclerosis, but not a method for production or purification of ApoE.
Japanese Patent Application No. JP 61096998 assigned to Mitsubishi Chemical Industries Ltd. described the cloning and expression of apolipoprotein A-1 analog.
Japanese Patent Application No. JP 60163824 assigned to Nippon Shinyaku KK described the use of serum lipoproteins including ApoE as drug carriers for intravenous injection. The ApoE in JP 60163824 is not derived from recombinant sources.
Co-assigned, copending patent applications U.S. Ser. No. 896,750, filed on Aug. 14, 1986 and U.S. Ser. No. 085,651, filed on Aug. 14, 1987 (a CIP of U.S. Ser. No. 896,750) disclosed methods directed to small scale purification of ApoE. While the recombinant ApoE analogs produced by these methods were very pure, it was necessary to develop a suitable method for industrial production scale-up. Further, it was desirable to produce an ApoE analog containing lower endotoxin levels than those previously produced. Specifically, the method disclosed in U.S. Ser. No. 896,750 produces an ApoE analog which is greater than 90% pure but has an endotoxin level in the range of 500,000-1,000,000 pg/mg.
The method described in U.S. Ser. No. 085,651 utilizes a urea solution throughout and involves batch chromatography on Phenyl-SEPHAROSE.TM., Heparin-SEPHAROSE.TM. and DEAE-SEPHAROSE.TM. (SEPHAROSE.TM. is a trademark for a specific cross-linked agarose matrix) columns. The resulting ApoE analog is greater than 95% pure and contains less than 2,000 pg endotoxin per mg. The use of urea necessitated the performance of the chromatography steps at 4.degree. C.-10.degree. C. because urea is unstable and the ApoE analog tends to degrade and aggregate in the presence of urea unless the experiments are performed in low temperatures.
Additionally, the chromatography methods in U.S. Ser. No. 085,651 are batch methods involving overnight stirring, conditions which make scale-up very difficult. The endotoxin level of the resulting analog is still high.
The novel methods described herein, i.e. Scheme I and Scheme II, use Triton X-100 (Triton.sup.R) to protect ApoE from degradation and aggregation. Triton.sup.R allows the ultrafiltration and column chromatography step to be performed at room temperature. In addition, no batch steps are required in this process. The methods are therefore suitable for scale-up.
Furthermore, Triton.sup.R enables the use of acidic pH conditions; this is a distinct advantage since acidic pH conditions in the absence of Triton.sup.R lead to precipitation of the ApoE analog. The addition of Triton.sup.R allows the enrichment of ApoE by an acidic pH extraction step and also the use of cation exchange chromatography.
The ApoE analog produced by the methods disclosed herein is extremely pure and has a very low endotoxin level, i.e. about 25 pg/mg; therefore, the resulting ApoE analog can be used for animal trials. We have shown that the resulting ApoE analog can be lyophilized and still retains its biological activity on subsequent dissolution, and we have demonstrated that the lyophilized ApoE is very stable.